The objective of this study was to determine whether methylcobalamin inhibits the ototoxic action of gentamicin. Fourteen pigmented guinea pigs were randomly divided into experimental and control groups. Each animal in the experimental group was given a large single dose of gentamicin (125 mg/kg i.m.), followed by immediate administration of methylcobalamin (1 mg kg i.m.). In the control group, the animals received gentamicin only, at the same dose as the experimental group. The compound action potentials (CAPs) in the right ears of animals were measured using a signal processor before and 15, 30, 45, 60 and 75 min after injection of the drugs. The sound stimulus was presented by means of clicks and tone pips at 2, 4 and 8 kHz. Following the electro-physiological tests, the animal was sacrificed and prepared for scanning electron microscopic observation. There was prolongation of latency and diminution of amplitude of CAPs N1 and N2 15 min after injection of gentamicin. Although the N1-N2 intervals increased as the time after injection of gentamicin increased, the variations were still within normal ranges. In contrast, there was no prolongation of N1 latencies or N1-N2 intervals 30 min after injection in the experimental group. There were no marked changes in N1 or N2 amplitudes, compared with the results before injection, in the experimental group. The firing mechanism of the inner ear may be affected by a single dose of gentamicin. This suggests that the transient ototoxic action of gentamicin hardly affected the conductive function of the nerve fibres, even though the firing function of the inner ear was influenced. Methylcobalamin may inhibit the ototoxic side effects of gentamicin.